This invention relates to the development of antigens suitable as vaccines for preventing whooping-cough.
Whooping-cough is a severe, highly contagious respiratory disease resulting from infection with the bacterium Bordetella pertussis. At the present time there is no fully effective treatment, the disease is associated with substantial morbidity and mortality, and is widespread throughout the world. Whooping-cough is particularly severe in infants.
Sato et al. 46 Infect. & Immunity 422, 1984 describe the use of whole-cell vaccines for controlling whooping cough, and their recent replacement by defined vaccines:
Pertussis vaccine composed of killed cells has been playing a role in the reduction of whooping cough for more than 40 years throughout the world. At the same time, however, it is one of the most rejected of vaccines because of its adverse reactions. It is now time that the whole-cell vaccine be replaced by a more defined vaccine that is composed of specific components and is able to have its protective potency evaluated by means of purified reference protective antigens or antibodies. In Japan, a pertussis vaccine in use since 1981 has had some adverse effects reduced by removal of the endotoxin from a fraction of culture supernatant of Bordetella pertussis phase I cells and inactivation of some of the toxicity with Formalin. The main components of the vaccine are formalinized pertussis toxin (PT) and filamentous hemagglutinin (FHA). Now we understand that PT is the most potent antigen and FHA is a helpful protective antigen and that their antibodies play an important role in protecting mice from infection and disease caused by the pathogen. PA1 PT is a heterohexameric protein consisting of five subunits, designated S.sub.1 to S.sub.5 Based on their gene sequences, the molecular weights of the subunits are 26,024 for S.sub.1, 21,925 for S.sub.2, 21,873 for S.sub.3, 12,058 for S.sub.4, and 11,013 for S.sub.5. The largest of the subunits, S.sub.1, is responsible for ADP-ribosylating the .alpha. subunits of a family of homologous, guanine nucleotide-dependent regulatory complexes (designated G or N) in eucaryotic cells. The other four subunits are thought to form a pentameric base unit composed of two dimers (S.sub.2 S.sub.4 [dimer 1] and S.sub.3 S.sub.4 [dimer 2] which are connected to each other by the S.sub.5 subunit. The function of the base structure is in binding to host cell receptors and providing a means for the S.sub.1 subunit to penetrate the cytoplasmic membrane. [Citations omitted.] PA1 The S1 subunit is one of the few proteins that does not contain lysine residues. This observation has important implications for the development of a new vaccine against pertussis, since normally for vaccine preparation, bacterial toxins are detoxified with chemicals that react mainly with lysine residues. Accordingly, we have observed that the detoxification of PT requires more severe conditions that those used for the other bacterial toxins and that following treatment with glutaraldehyde, S2, S3, S4 and S5 are crosslinked and form aggregates of high molecular weight, while S1 retains its original size. PA1 The cloned and sequenced pertussis toxin gene will facilitate the development of an efficient and safer vaccine against whooping cough. By comparison to other toxin genes with similar biochemical functions, and by physical identification of the active sites either for the ADP-ribosylation in the S1 subunit or the target cell binding in subunits S2 through S4, it is now possible to modify those sites by site-directed mutagenesis of the B. pertussis genome. These modifications could abolish the pathobiological activities of pertussis toxin without hampering its immunogenicity and protectivity. Alternatively, by knowing the DNA sequence it will be possible to map protective epitopes. Synthetic oligopeptides that include those epitopes also will be useful in the development of a new generation of vaccines.
To prepare such defined vaccines PT and FHA are purified by successive column chromatography procedures. Sato et al., Id. The PT and FHA is then treated with 0.2% formalin at 39.degree. C., and with 0.1% formalin two more times every other day, prior to dialysis. Id. at 415.
An alternative procedure involves preparation of PT free of FHA, and subsequent treatment of PT with 0.05% glutaraldehyde to yield detoxified PT, which is effective in protecting mice from infection. Munoz et al. 32 Infect & Immun. 243, 1981.
The terminology used to define pertussis toxin is confused in the literature. PT is "also known as lymphocytosis-histamine-sensitizing factor, islet-activating protein, and pertussigen." Locht et al. 232 Science, 1258, 1986. Recently Armstrong et al. 55 Infect & Immun., 1294, 1987 have described PT as follows:
Serine, threonine, and tyrosine residues are represented more frequently in PT than in average E. coli proteins. (Locht et al. Science 232: 1258, 1986.) It is thought that the hydroxyl groups of these residues may be involved in the quaternary structure of PT, through hydrogen bonding. Locht et al., Id. There are no lysine residues in the S.sub.1 subunit of the PT gene which "may explain why lysine-specific chemical modification do not affect the hydroxyl and enzymatic activities of S1." Locht et al., Id.
Nicosia et al., 83 Proc. Nat. Acad. Sci. U.S.A. 4631, 1986, note that harsh conditions for inactivation of PT are required because of the shortage of lysine residues in subunit S1.
PT is described as sensitive to iodination, but such sensitivity can be reduced by adsorption to fetuin-agarose. Armstrong et al. supra. One possible reason for this sensitivity is the large number of tyrosine residues in PT, some of which may be critical to its functions as a toxin. In conventional iodination research, the modification of such important tyrosines could be one explanation for loss of activity. Armstrong et al. Id.
Recently, the cloning of DNA encoding PT subunits has been described. Locht et al. supra; Nicosia et al., supra. Locht et al. note the use of such a gene for developing vaccines: